Dergez Timea, Lorinczy Dénes, Könczöl Franciska, Farkas Nelli, Belagyi Joseph
Institute of Bioanalysis Faculty of Medicine, H-7624 Pécs, Hungary.
BMC Struct Biol. 2007 Jun 24;7:41. doi: 10.1186/1472-6807-7-41.
Thermal denaturation experiments were extended to study the thermal behaviour of the main motor proteins (actin and myosin) in their native environment in striated muscle fibres. The interaction of actin with myosin in the highly organized muscle structure is affected by internal forces; therefore their altered conformation and interaction may differ from those obtained in solution. The energetics of long functioning intermediate states of ATP hydrolysis cycle was studied in muscle fibres by differential scanning calorimetry (DSC).
SETARAM Micro DSC-II was used to monitor the thermal denaturation of the fibre system in rigor and in the presence of nucleotide and nucleotide analogues. The AM.ADP.Pi state of the ATP hydrolysis cycle has a very short lifetime therefore, we mimicked the different intermediate states with AMP.PNP and/or inorganic phosphate analogues Vi and AlF4 or BeFx. Studying glycerol-extracted muscle fibres from the rabbit psoas muscle by DSC, three characteristic thermal transitions were detected in rigor. The thermal transitions can be assigned to myosin heads, myosin rods and actin with transition temperatures (Tm) of 52.9 +/- 0.7 degrees C, 57.9 +/- 0.7 degrees C, 63.7 +/- 1.0 degrees C. In different intermediate states of the ATP hydrolysis mimicked by nucleotide analogues a fourth thermal transition was also detected which is very likely connected with nucleotide binding domain of myosin and/or actin filaments. This transition temperature Tm4 depended on the mimicked intermediate states, and varied in the range of 66-77 degrees C.
According to DSC measurements, strongly and weakly binding states of myosin to actin were significantly different. In the presence of ADP only a moderate change of the DSC pattern was detected in comparison with rigor, whereas in ADP.Pi state trapped by Vi, AlF4 or BeFx a remarkable stabilization was detected on the myosin head and actin filament which is reflected in a 3.0-10.0 degrees C shift in Tm to higher temperature. A similar effect was observed in the case of the nonhydrolyzable AMP.PNP analogue. Differential DSC measurements suggest that stabilization actin structure in the intermediate states of ATP hydrolysis may play an additional role in actin-myosin interaction.
热变性实验得到扩展，以研究横纹肌纤维中天然环境下主要运动蛋白（肌动蛋白和肌球蛋白）的热行为。在高度有序的肌肉结构中，肌动蛋白与肌球蛋白的相互作用受内力影响；因此，它们构象和相互作用的改变可能与在溶液中得到的情况不同。通过差示扫描量热法（DSC）研究了肌肉纤维中ATP水解循环长时间功能中间态的能量学。
使用SETARAM Micro DSC-II监测纤维系统在僵直状态以及存在核苷酸和核苷酸类似物时的热变性。ATP水解循环的AM.ADP.Pi状态寿命非常短，因此，我们用AMP.PNP和/或无机磷酸盐类似物Vi和AlF4或BeFx模拟不同的中间态。通过DSC研究兔腰大肌甘油抽提的肌肉纤维，在僵直状态下检测到三个特征性热转变。这些热转变可归因于肌球蛋白头部、肌球蛋白杆和肌动蛋白，转变温度（Tm）分别为52.9±0.7℃、57.9±0.7℃、63.7±1.0℃。在用核苷酸类似物模拟的ATP水解不同中间态中，还检测到第四个热转变，其很可能与肌球蛋白的核苷酸结合结构域和/或肌动蛋白丝有关。该转变温度Tm4取决于模拟的中间态，在66 - 77℃范围内变化。
根据DSC测量，肌球蛋白与肌动蛋白的强结合态和弱结合态有显著差异。与僵直状态相比，仅存在ADP时，DSC图谱仅有适度变化，而在用Vi、AlF4或BeFx捕获的ADP.Pi状态下，在肌球蛋白头部和肌动蛋白丝上检测到显著的稳定化，这表现为Tm向更高温度偏移3.0 - 10.0℃。在不可水解的AMP.PNP类似物情况下也观察到类似效应。差示DSC测量表明，ATP水解中间态中肌动蛋白结构的稳定化可能在肌动蛋白 - 肌球蛋白相互作用中起额外作用。
